1. Field of the Invention
This invention relates to a method of measuring lipid-bound sialic acid (LSA) and a LSA separating agent for the same.
2. Description of the Prior Art
Recently, it has been reported that when persons suffer from cancer, LSA concentration in their blood increases. Regarding this increase in LSA concentration, many reports have been made. It has also been reported that even in a patient having primary gastric cancer whose total sialic acid concentration has not increased yet, LSA concentration of the patient has significantly increased (Oki, S., J. Jpn. Soc. Cancer Ther., vol 18, pp 692-703 (1983)).
As method of measuring LSA, the method developed by N. Katopodis et al. (Res. Commun. Clin. Path. Pharm., vol. 30, pp 171-180 (1980)) has widely been employed. This method comprises the following steps. First, each serum is pipetted into a test tube and distilled water is added. The mixture is cooled to 4.degree. C. and an extractant (chloroform : methanol=2:1) is added. This mixture is vigorously stirred for 30 seconds and centrifuged at room temperature. The supernatant is put into another test tube and an aqueous phosphotungstic acid solution is added to precipitate LSA. The precipitate is collected by centrifuging and dissolved in distilled water. A resorcinol reagent is added to the solution and allowed to react at 100.degree. C. for 20 minutes. After being cooled to room temperature, a solvent mixture of butyl acetate-butanol is added to extract a colored material. Absorbance of the butyl acetate-butanol layer is measured to determine LSA concentration of the sample.
Since the method of Katopodis et al. is complicated as mentioned above, an enzyme method has been developed (Japanese Patent KOKAI No. 60-78597). In this method, after pretreatments of a sample, such as dilution and solvent extraction of free fatty acids etc., the sample is divided into two parts. One part is kept for measuring total sialic acid, and phosphotungstic acid is added to the other part to precipitate LSA. The sialic acid concentration of the part not treated with phosphotungstic acid and the supernatant of the other part treated with phosphotungstic acid are measured by the enzyme method such as developed by Sugahara et al. (Clin. Chim. Acta, vol. 108, pp 493-493 (1980)) where neuraminidase and N-acetylneuraminic acid aldolase are employed. LSA concentration of the sample is determined by subtraction of the above two sialic acid concentrations. This method is simpler than the previous method, but in this method, LSA concentration is indirectly determined by subtraction.